Controlled cell death is critical for the life of a human; too much cell death can cause the symptoms of cystic fibrosis and also lead to diseases such a neurodegeneration and acquired immune deficiency syndrome (AIDS). In contrast, too little cell death can lead to cancer or autoimmune diseases. Recent studies have defined the pathway of cell death as “apoptosis” and have identified some of the biochemical steps involved.
Apoptosis is a homeostatic mechanism involved in the controlled death of obsolete cells during metamorphosis, differentiation, cell turnover, and hormone mediated deletion of thymocytes (Wyllie et al. Int. Rev. Cytol. 1980 68:251-306). Apoptosis has also been identified as the mechanism of cell killing during growth factor withdrawal (Rodriguez-Tarduchy et al. EMBO J. 1990 9:2997-3002; McConkey et al. J. Biol. Chem. 1990 265:3009-3011), T-cell deletion, treatment with many cytotoxic agents (Cohen, J. J. and Duke, R. C. J. Immunol. 1984 132:38-42; Barry et al. Biochem. Pharmacol. 1990 40:2353-2362; Kaufmann, S. H. Cancer Res. 1989 49:5870-5878; and McConkey et al. Science 1988. 242:256-259), and following hyperthermia (Barry et al. Biochem. Pharmacol. 1990 40:2353-2362; Lennon et al. Biochem. Soc. Trans. 1990 18:343-345; Takano et al. J. Pathol. 1991 163:329-336).
Central to the mechanism of apoptosis is internucleosomal DNA digestion by endogenous endonucleases. Mammalian cells contain a variety of endonucleases which could be involved in internucleosomal DNA digestion. It was originally postulated that the primary endonuclease involved in apoptosis is a Ca2+/Mg2+-dependent endonuclease. Several Ca2+/Mg2+-dependent endonucleases have been identified, one of which is deoxyribonuclease I (DNase I), (Peitsch et al. EMBO J. 1993 12:371).
Recent experiments, however, indicate that DNase I may not be the primary endonuclease involved in apoptosis. It has been found that many cells do not contain this endonuclease. The role of DNase I, or any other Ca2+/Mg2+-dependent endonuclease is further unlikely, as often no increase or only a minor increase in Ca2+ levels occurs in apoptotic cells (Eastman, A. Cell Death and Differentiation 1994 1:7-9).
An alternate endonuclease that is active below pH 7.0 and has no apparent requirement for Ca2+ or Mg2+ has been detected (Sorenson et al., J. Natl Cancer Inst. 1990 82:749). This alternate endonuclease was identified as deoxyribonuclease II (DNase II; Barry, M. A. and Eastman, A. Archives of Biochem and Biophys. 1993 300(1):440-450). It was proposed that this enzyme is involved in the internucleosomal digestion or fragmentation of DNA which is one of the early steps in the pathway of apoptosis. Another report that has implicated DNase II in cell death involves lens fiber cell differentiation, a process where the cells lose their nuclei in a manner similar to apoptosis (Torriglia, A. et al. 1995 J. Biol. Chem. 270:28579-28585). In this process, the chromatin condenses and the cells degrade their genomic DNA. DNase II was found by immunocytochemistry to be localized in the cytoplasm but translocated to the nucleus of the fiber cell before degeneration. These findings implicate DNase II as the endonuclease responsible for genomic degradation observed during lens nuclear degeneration, and further support a role for this enzyme in mechanisms of cell death.
However, more recent results have implicated yet another endonuclease, referred to CAD or caspase-activated deoxyribonuclease, in apoptosis (Enari, M. et al. 1998 Nature 391:43-50). Thus, it remains to be determined which specific endonuclease is involved in apoptosis,
The enzyme referred to herein as deoxyribonuclease IIα (DNA IIα) was isolated and purified and the amino acid sequence determined (PCT/US97/18262). The DNA sequences for both the human and bovine proteins of DNase IIα have also been cloned (PCT/US97/18262). Use of DNA IIα in alleviating the suffering in patients with cystic fibrosis is also disclosed in this PCT application.
In cystic fibrosis, the lungs of patients fill with the remnants of dead cells, and in particular with the DNA from these dead cells. The presence of DNA makes the mucous plugs too viscous to expel. A suggested therapy for these symptoms is the use of DNase I to digest the DNA, thereby permitting expulsion of the mucous plugs. However, this therapy has not been particularly effective due to inactivity of the DNase I enzyme in the presence of actin, also present in the sputum.
It is believed that DNase II enzymes and variations thereof may provide a more effective therapeutic alternative.
Another isoform of the DNase II enzyme, referred to herein as deoxyribonuclease IIβ (DNase IIβ) has now been identified and the gene and protein sequences for the mouse and human homolog have been determined.